The critical role of regulatory T cells (Tregs) in the regulation of immune responses to foreign, self, and tumor- associated antigens has sparked considerable interest in the development of strategies for the manipulation of Tregs for the treatment of autoimmunity and cancer. Despite substantial progress in the field, a critical gap in knowledge remains. Although substantial evidence is consistent with the hypothesis that thymic-derived Tregs recognize self antigens, the identity and nature of these self antigens remain unknown. Continued existence of this gap remains an important problem because many aspects of Treg biology are highly dependent on antigen recognition, including development, peripheral homeostasis, anatomical distribution, activation status, and suppressive function. Thus, the lack of knowledge regarding the identity and nature of Treg antigens has restricted progress in the field and limited the ability to selectively expand or deplete antigen-specific Tregs for the treatment of autoimmunity and cancer. The long-term goal is to understand the development, antigen specificity, and function of Foxp3+ Tregs in the maintenance of immune tolerance and the modulation of cancer progression. The objective / specific aim of this proposal is to identify the antigen recognized by an endogenous population of prostate-specific Tregs, termed MJ23 Tregs. The central hypothesis is that MJ23 Tregs recognize a prostate-associated self peptide derived from a protein encoded by Autoimmune regulator (Aire)-dependent transcripts in the thymus. This hypothesis is based on previously published data from the applicant's laboratory. To achieve the objective, three complementary approaches will be utilized: 1) screening of candidate proteins encoded by Aire-dependent, prostate-specific transcripts in the thymus; 2) screening of antigens encoded by a prostate-derived cDNA expression library; 3) biochemical identification of the antigen from fractionated extracts isolate from primary prostate tissue. The proposal is made possible by use of an innovative model system that provides both a reproducible source of antigen enriched at a distinct anatomical site (prostate tumors) and a T cell probe for identifying this antigen. The proposed work is significant because identification of the antigen will bridge critical gaps in knowledge and open new areas of investigation that are not currently possible. These areas include: 1) analysis of endogenous, antigen-specific Tregs using peptide- MHC multimer reagents in the context of organ-specific tolerance, inflammation, and cancer; 2) pre-clinical research for the treatment of autoimmunity by expansion of prostate-specific Tregs via administration of exogenous antigen; 3) studies in which the function, trafficking, and homeostasis of MJ23 Tregs can be analyzed via regulated transgenic overexpression or conditional deletion of the antigen; 4) biophysical elucidation of the affinity and kinetics of MJ23 TCR binding to the antigenic ligand, and determination of the crystal structure of the ternary complex.